We have previously demonstrated that both CD8+ and CD4+ T lymphocytes derived from cancer patients can manifest MHC-restricted recognition of tumor associated antigens. This has been shown for patients with melanoma, colon carcinoma, breast carcinoma, and lymphoma. Current projects focus on identifying melanoma-associated proteins and peptides recognized by CD4 T cells, as well as extending this work to prostate cancer. These studies are directly relevant to clinical efforts to develop cancer vaccines. l. Identification of tyrosinase peptides recognized by CD4+ T cells. We recently showed that tyrosinase, a protein with expression linked to the melanocytic linage, was recognized by melanoma-reactive CD4 T cells from one patient. Recognition was MHC class II-restricted. By testing overlapping 15-mer peptides derived from tyrosinase, we have identified 2 peptides which evoke specific T cell recognition. Both are restricted by HLA-DRbeta1*O4O1, and both are based on the non-mutated gene sequence. These peptides are being used to raise melanoma- reactive PBL from other patients. 2. Search for other melanoma-associated proteins recognized by CD4+ T cells from melanoma patients. We have cultured and characterized CD4 T cells specifically recognizing unidentified melanoma epitopes. Two strategies are under study for epitope characterization. One approach uses protein separation techniques to fractionate detergent lysates of melanoma cells, followed by sequencing the bioreactive fractions. A partial protein purification has thus far been achieved in one melanoma system. The second approach, recently begun, is based on novel molecular cloning strategies in collaboration with the laboratory of Dr. Drew Pardoll. 3. Defining antitumor immune responses in prostate cancer patients. Studies of antitumor immunity in prostate cancer are rare, in part owing to difficulties in obtaining adequate amounts of tumor tissues for testing. We have developed a highly successful method for establishing long term prostate epithelial cell lines from fresh specimens, involving immortalization with the HPV E6 and E7 proteins. These tumor cells are being used to culture prostate cancer-reactive PBL from a number of patients. In addition, MHC class I-restricted peptides based on the prostate-associated proteins PSA and PAP are being used to raise reactive CD8+ PBL from patients with appropriate ALA types. To date, one stimulatory PAP peptide, restricted by HLA-II, has been identified.